Sphingolipid’Cholesterol Rafts Diffuse as Small Entities in the Plasma Membrane of Mammalian Cells

Author :A. Pralle, P. Keller, E.-L. Florin, K. Simons, and J.K.H. Hörber
Publication :Journal of Cell Biology
Publisher :The Rockefeller University Press
Volume :148
Number :5
Pages :997
Year :2000

To probe the dynamics and size of lipid rafts
in the membrane of living cells, the local diffusion of
single membrane proteins was measured. A laser trap
was used to confine the motion of a bead bound to a
raft protein to a small area (diam
# 100 nm) and to
measure its local diffusion by high resolution single particle
tracking. Using protein constructs with identical
ectodomains and different membrane regions and vice
versa, we demonstrate that this method provides the
viscous damping of the membrane domain in the
lipid bilayer. When glycosylphosphatidylinositol (GPI)
-anchored and transmembrane proteins are raft-associated,
their diffusion becomes independent of the type
of membrane anchor and is significantly reduced compared
with that of nonraft transmembrane proteins.
Cholesterol depletion accelerates the diffusion of raftassociated
proteins for transmembrane raft proteins to
the level of transmembrane nonraft proteins and for
GPI-anchored proteins even further. Raft-associated
GPI-anchored proteins were never observed to dissociate
from the raft within the measurement intervals of
up to 10 min. The measurements agree with lipid rafts
being cholesterol-stabilized complexes of 26
6 13 nm in
size diffusing as one entity for minutes.